Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.
Cyclin D1-cdk4 complexes promote the G0/G1 phase transition, and as such their activity is tightly regulated by a variety of mechanisms, including the transcription and translation of the mitogen sensor cyclin D1 and positive and negative regulatory phosphorylation of cdk4 (1,2). The most well-characterized substrate of cyclin D-cdk4 is the G1 gatekeeper, Retinoblastoma (Rb), and deregulation of cdk4 potentially accelerates Rb phosphorylation and cell cycle transitioning, promoting cancer development (3). Cyclin D1 and cdk4 are overexpressed in a variety of human cancers, and in mouse models, loss of either cdk4 or cyclin D1 prevents the development of certain oncogene-driven tumors, further evidence of their involvement (4-6). However, the levels of cyclin D or cdk4 in a tumor may not be reliable measures of activity, due to the fact that a third protein, an assembly factor such as p27Kip1 or p21Cip1, is required both for the stabilization and then the subsequent activation of this complex (1, 7).
Independent of its ability to assemble cyclin D-cdk4 complexes, p27 acts as a bona fide “switch” turning cyclin D-cdk4 complexes on or off, which in turn modulates cell cycle entry or exit (8, 9). Tyrosine (Y) phosphorylation of p27 on residues Y74, Y88 and Y89 opens the cyclin D-cdk4-p27 ternary complex, rendering it able to phosphorylate substrates such as Rb (9-14). Cyclin D-cdk4-p27 complexes isolated from cells in G0 lack Y phosphorylation on p27 and are catalytically inactive, while complexes isolated from proliferating cells are Y phosphorylated and active. Y88 and Y89 are part of the 3-10 helix, which has been shown to insert into the ATP binding cleft in cdks (15). When not phosphorylated, residues Y88/Y89 sequester within this binding pocket and block cdk4 activity (p27 switched OFF). NMR and other studies suggest phosphorylation of Y88/Y89 induces a conformational change in p27, ejecting the Y88/Y89 loop, opening the cyclin D-cdk4 complex, permitting both ATP access and the required phosphorylation on cdk4 residue T172 by the Cyclin Activating Kinase (CAK), the latter causing activation of cdk4 (p27 switched ON) (11, 12, 14, 16). Thus, p27's control of cyclin D-cdk4 makes it a key player in the regulation and integration of a cell's response to extracellular signals.
Members of the Src Family of Kinases (SFKs), including Src, Yes, and Lyn have been shown to phosphorylate p27 in vitro (9). Moreover, distantly related kinases, such as the Abelson kinase Abl and the Janus kinase, Jak2, also appear competent to phosphorylate p27 (11, 12, 17). The Src kinase family consists of 8 members: Src, Yes, Fyn, Fgr, Lyn, Hck, Lck, and Blk (18). Frk, Srm, Src42A and PTK6/Brk comprise a distantly related, but distinct family (19, 20). Brk is an intracellular tyrosine kinase expressed in normal epithelial cells and overexpressed in 60% of breast cancers. Brk has been shown to phosphorylate p27 in vitro and in vivo, and studies have shown that Brk is a higher affinity binder than members of the SRK family. Knockdown of Brk in breast cancer cells also prevents p27 phosphorylation, even in the presence of Src and other SFKs, suggesting that it is the physiological kinase for p27 Y phosphorylation (14). All of these kinases share a common domain organization comprising the tyrosine kinase domain (also termed SH1), as well as one each of the protein-protein interaction modules SH2 and SH3, which bind to phosphotyrosine and proline-rich sequences (PxxP), respectively. The SH2 and SH3 domains recognize specific amino acid sequences within the SFK itself, thus adopting an autoinhibited state. Upon release from this inhibition by upstream signalling molecules, the SH2 and SH3 domains are free to bind downstream SFK target proteins (21).
The principal task of the cell cycle is to ensure that a cell's DNA is faithfully duplicated and evenly distributed to daughter cells. Loss of control over this process is a hallmark of cancer. Indeed, as mentioned above, direct perturbation of most genes involved in cell cycle control has been observed in human cancers. Cell cycle transitions are tightly controlled by the actions of the cyclin-cdks. New therapeutic compounds, which modulate these actions should prove effective in the treatment of hyperproliferative disorders, including malignant disease.